Dengue virus serotypes 1-4 (DENV-1-4) and several other insect-borne flaviviruses, including Japanese encephalitis virus (JEV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV) are important human pathogens. The humoral immune response is an essential part of host protection against these flaviviruses. Monoclonal antibodies (MAbs) from humans or nonhuman primates are a valuable tool to study the antigenic structures of the major protective antigens and mechanisms of neutralization, and to explore passive immunization as an alternative to vaccines for prevention of infections. We have studied a number of flavivirus-reactive MAbs from chimpanzees recovered by repertoire cloning, including DENV-4-specific MAb 5H2, DENV cross-reactive MAb 1A5, JEV-specific MAbs A3, B2 and A3. JEV-specific MAbs neutralize the virus at a high titer in vitro and protect against infection using a mouse model. MAb 5H2 has also been shown to protect against DENV-4 infection when passively administered to rhesus monkeys as human surrogates. MAb 5H2 neutralizes the virus by binding to domain I (DI) of the envelope glycoprotein (E) with nanomolar affinity. We have now determined the crystal structure of the DENV-4 E ecto-domain (amino acid residues 1-395) in complex with the Fab fragment of 5H2 at 3.2 resolution as well as the 2.7 resolution structure of a smaller complex including only the DI and Fab. The antibody-antigen contacts involve DENV-4 DI residues Glu172, Lys174, Pro176, Asp177, Glu180 and DI-DIII linker residue Arg293. Residues Lys174 and Pro176 have been identified as Fab 5H2 epitope determinants by sequencing of neutralization-escape mutants. The antibody-antigen contacts explain the serotype specificity of 5H2. Modeling by positioning the DENV-4 DI/Fab 5H2 crystal structure onto the DENV-2 E post-fusion trimer shows that MAb 5H2 interferes with fusogenic conformational change of DENV-4 E. Of interest is the antibody binding interaction of Arg293 in the DI/DIII linker region which participates in the dimer-to-trimer shift during membrane fusion, suggesting that MAb 5H2 might interfere with this event. A search of human homologues to the chimpanzee 5H2 variable domains reveals that an antibody with variable domains sharing >98% sequence homology with those of 5H2 could be produced from human germ-line genes. In light of the structural analysis, such human antibodies binding to the corresponding portion of other three DENV serotypes may represent important effectors of serotype-specific human humoral immunity to DENV. Antibody-dependent enhancement (ADE) has been proposed as an underlying pathogenic mechanism of severe dengue characterized by DHF/DSS. ADE occurs because pre-existing neutralizing antibodies and infecting DENV form complexes that bind to Fc receptor-bearing cells, leading to decreased antibody neutralizing capacity and increased viral replication. We have shown that cross-reactive MAb 1A5 (IgG1 subclass) mediates ADE in monocytes and in monkeys and a 9-amino acid deletion in the Fc region ablates its ADE activity in vitro. MAb 1A5 with the key IgG2- or IgG4-specific substitution shows Fc&#947;R binding properties of IgG2 or IgG4. These IgG 1A5 subclasses together with mouse-human chimeric DENV or WNV Mabs containing human Fc were employed to analyze their neutralizing activities and Fc&#947;R binding interactions using CV-1 cell transfectants displaying functional Fc&#947;RIA or RIIA and control CV-1 cells. The results showed DENV neutralization by IgG1, IgG3 and IgG4 MAbs was enhanced in high-affinity Fc&#947;RI cells and diminished in low affinity Fc&#947;RII cells. In FcR-negative cells, IgG3 was the strongest neutralizer, and IgG 2 the weakest. Thus, DENV neutralization is modulated by the Fc region in an IgG subclass dependent manner. The IgG antibody subclass response to DENV infection or vaccination may influence the neutralizing capacity. Fabs were also recovered from a TBEV-infected chimpanzee. Among these, Fab 2E6 neutralized TBEV most efficiently and was selected for further characterization. A sequence search of the Fab 2E6 VH and Vk variable segments identified homologous human germ line gene segments. Humanized MAb 2E6 neutralized TBEV at 0.02-0.04 &#956;g/ml, similar to the tiers of highly-neutralizing MAbs against other flaviviruses. The major epitope determinant of MAb 2E6 was localized to Lys202 in E domain I by sequencing of neutralization escape mutants and verified by loss of binding to the recombinant E variant. Titration of MAb 2E6 against intracerebral (ic) infection with 50 lethal dose50 (LD50) of TBEV/DENV-4 24 hr later showed a dose-dependent response in terms of the survival rate. The 50% protective dose of MAb 2E6 was calculated at 18 g per mouse. The protective capacity of MAb 2E6 was also analyzed using three-week-old Swiss-Webster mice against intra-peritoneal challenge with the TBEV-related LGTV. The peripheral route of LGTV challenge is relevant to TBEV infection in humans. Using the mouse model, the 50% protective dose of MAb 2E6 against challenge with 50 LD50 of LGTV was 0.24 g per mouse or lower. Results also showed that MAb 2E6 administered at a single dose of as low as 1.2 g protected mice against prior LGTV infection significantly, as measured by average survival time. These experiments demonstrate proof of concept for the therapeutic potential of MAb 2E6 against TBEV infection.